Adipose tissue-derived stem cells (ADSC) represent a large sub-population of cells isolated from the stromal vascular fraction of collagenase-digested adipose tissue [11
]. ADSC are comparable to bone marrow and umbilical cord blood derived mesenchymal stem cells with respect to differentiation capacity, immune phenotype, and morphology [11
]. ADSC possess multi-lineage potential when induced under lineage-specific conditions in-vitro [11
]. Their pluripotency has been utilized to repair muscle tissues and improve wound vascularization [19
In our present work, we have shown that unmodified autologous ADSC injected into the penis of impotent type-II diabetic rats are associated with significant functional improvement in erectile function, as compared to untreated animals (p<0.002). Intracorporal pressures improved and approached, but did not equal, normal values. This could be due to a variety of factors: erectile function was assessed only 3 weeks after injection, and this may have been insufficient time for maximal treatment effect. Also, the diabetic state remained uncontrolled throughout, and this could have directly damaged and/or countered the effects of the ADSC following transplantation. Results of immunohistochemistry and real-time PCR studies together suggest that the treatment effect may be mediated at least in part by decreased intracorporal tissue apoptosis of the treated animals and increased number of sinusoidal endothelial cells. Interestingly, only relatively few BrdU labeled ADSC were visualized within the penis, suggesting that the principal mechanism of effect from the ADSC may not be through direct transformation into local cell types, but through the elaboration of cytokines, growth factors affecting cell surface receptors, and indirect changes within the extracellular compartment of local tissue.
Real-time PCR showed significantly lower expression of Caspase-3 in treated versus control group animals (p
<0.05). Immunohistochemical staining for TUNEL was similarly lower in ADSC treated animals versus control group animals (p
<0.05). While the exact mechanisms for these findings are unclear, it is possible that increased local neovascularity and intracorporal endothelial cell count, mediated by ADSC, improved local tissue health and function. Paracrine secretion of cytokines and growth factors has been observed in other studies with mesenchymal stem cells, and it is possible that ADSC secretion of such factors improved tissue health in treated animals [21
Staining for nNOS was compared using the dorsal penile nerves, rather than a sampling of intracorpopral tissue, because quantification of nNOS staining within intracorporal cavernosal nerves is less reliable [22
]. Dosal nerve nNOS is an acceptable surrogate for presence of cavernosal nNOS for two reasons: first, cavernous nerves travel with the dorsal nerve bundles to reach the distal-most extremities of the penis. Second, it has been shown that selective damage to the cavernous nerves results in decreased nNOS within both the intracorporal cavernous nerves and within the dorsal penile nerves [22
]. Cellek et al. showed that uncontrolled type-I diabetes results in progressive loss of nNOS in dorsal penile nerves [23
]. Early-phase decrease in axon (not cell body) nNOS occurred. This was reversible with administration of insulin and resulted in decreased apoptosis. Axon and cell body structural damage occurred together in a later, non-reversible phase [23
]. Because ADSC treatment was associated with increased nNOS, it is possible that ADSC mediated a rehabilitative effect on nitrergic neuron axons and ganglia. Also, it is possible that improved physiologic erectile function provides improved inflow of oxygenated blood and growth factors, which would improve corporal tissue and dorsal nerve nNOS and decrease corporal tissue apoptosis. Serum testosterone and blood glucose levels were similar between groups, suggesting that treatment effect was not mediated by these factors.
A limitation of our work is that we could not assess the local retention or net survival of the injected ADSC. Emerging experimental imaging modalities that allow labeled cells to be followed in-vivo are crucial to our understanding of the stem cell molecular mechanisms and for the development of appropriate therapies and clinical trials. Another challenge to cellular therapy is how to improve local retention of the transplanted ADSC. Improved local retention could lead to greater improvement in erectile function after treatment. Use of bioabsorbable PLGA microspheres is a well-established means by which to improve local retention, survival, and possibly therapeutic effect of transplanted cells [24
]. Lastly, given that mesenchymal stem cells appear to spontaneously migrate to areas of injury [25
], it is essential that we study the behavior of ADSC not only in healthy animal models, but also in injured and diseased animal models, as homing factors, survival, dispersion, and differentiation characteristics may vary by disease status.
An additional limitation is the use of the BrdU label to identify the ADSC post-transplantation. Despite good cell labeling efficiency with BrdU, the longevity and specificity of the BrdU signal decreases substantially over time [27
]. More efficient and reliable stem cell labeling techniques are needed.
As a source of stem cells, adipose tissue has several key advantages: it is accessible by minimally invasive approaches (e.g. liposuction); harvest is generally associated with minimal morbidity; adipose tissue is self-replenishing; and, given its abundance in most people, it is likely that sufficient quantity for therapeutic applications could be harvested within a single procedure, thus precluding the need for cell culture. Freedom from reliance upon cell-culture has two important advantages. 1. There is intense debate concerning the perceived significant hazards associated with use of animal by-products in common culture media and cell-expansion protocols [28
]. Risk of transmission of viral, prion disease, and, other proteins that could initiate xenogenic immune responses has been reported [29
]. 2. From a regulatory perspective, omission of ex-vivo cell culture, which is considered a “modification” of the natural cell product, may facilitate regulatory approval for future clinical trials [31